STEWART LABORATORY
OLIGONUCLEOTIDE
LIGATION ASSAY FOR TELOMERIC G-rich 3’-overhang
(Adapted from Cimino-Reale
et al., NAR 2001, 29(7):e35)
PREPARE HIGH MOLECULAR WEIGHT DNA
We do several steps:
§
overnight at
37°C in the presence of proteinase K
§
1X Phenol
§
1X Phenol:Chloroform (1:1)
§
1X Chloroform
TREAT PREPARED HMW DNA WITH RNase (DNase-FREE)
§
Run treated DNA
out on a 1.0% gel to ensure that there is NO
RNA contamination (the presence of RNA causes many problems with this assay)
·
Phenol:Chloroform (1:1)
·
1X Chloroform
·
Re-suspend DNA
in ddH20 overnight at RT
TREAT 7 μg
OF DNA WITH Bal31 NUCLEASE
(this method should initially be validated by also treating
DNA with S1 nuclease, T7(Gene6) Exonuclease and Exonuclease III, and Exo I [Exo1
treatment needs to be done overnight and must be calibrated for the enzyme that
you are using. We’ve seen quite a bit of variation with this])
1.
Digest in 100 μL reaction (you want 3 units of Bal31 nuclease per
100 μL reaction)
2.
Digest at 30°C
for 15 min
3.
Phenol:Chloroform
4.
Chloroform
5.
Add 11 μL of 5M NaCl
6.
Add 300 μL 100% EtOH
7.
Spin 15 min at
4°C
8.
Wash with 70% EtOH
9.
Air dry
10.
Re-suspend in 12
μL ddH2O
11.
OD 2 μL of the DNA at
LABEL YOUR OLIGO
(this is for both the matched oligo, (CCCTAA)4, and the mismatched oligo,
(CCCTTA) 4, You will need 3.2 μL of oligo for each reaction). This is a sample reaction that
yields 50 μL (i.e. enough for 15 reactions)
§
Mix:
§
4 μL oligo at 2μM
§
5 μL 10x PNK buffer
§
15 μL ddH2O
§
24 μL gamma ATP (3000 Ci/mmol,
10mCi/mL)
§
2 μL PNK
40 min at
37°C
1.
Add
§
1 µL
0.1M cold rATP
§
1 μL PNK
20 min at 37°C
2.
Pass the labeled
oligo through a G25 column (2x)
3.
Check the specificity
(you want it to be 1 x 106 or higher)
PREPARE YOUR REACTIONS IN DUPLICATE
(GM847 DNA makes a great positive-control)
1.
The total
reaction volume is 20 μL
2.
Mix:
§
5 μg of RNA-free DNA and add ddH2O to 14.3 μL
§
2 μL 10X Taq ligase buffer
§
3.2 μL labeled oligo (you want
3.
Place the tube
at 50°C for 12-14 hr (use an oven, if you put it in a waterbath
it will evaporate)
4.
Quick spin the
tube and add 0.5 μL Taq
ligase
5.
Incubate another
5 hr at 50°C
6.
Add 80 μL ddH2O (containing ddH2O and
5M NaCl at a 7:1 ratio)
7.
Add 200 μL 100% EtOH
8.
Spin for 15 min
at max speed
9.
Wash with 70% EtOH (spin for another 5 min as the pellet often slips down
the side of the tube and allow to air dry)
10.
Re-suspend in 10
μL ddH2O and place at 50°C for 2 to 3
hours (or at 37°C, overnight)
PREPARE SAMPLES FOR GEL SEPARATION:
1.
Add 10 μl 2x denaturing DNA loading dye from Ambion
2.
Heat sample to
95oC for 5’ and crash on ice, quick spin
3.
OD your samples
(remember that the blank should have 1X loading dye that was heated in a
similar fashion to the samples)
4.
Aliquot equal
concentrations of DNA and equalize the volumes
5.
Heat sample to
95°C for 5 min and crash on ice, quick spin
6.
Load samples
onto a 5% Urea-TBE gel. We usually run
the first dye to about 1” from the bottom.
We run sequencing sized gels as the resolution on the smaller
gels isn’t good enough to do robust densitometry
7.
Dry down the
gels and expose (be sure to use the BioMax MS film
and the HE Transcreens at -70°C)
DILUTE SAMPLES TO 5 ng/μL
AND RUN A GAPDH PCR
(This insures that your ODs were
accurate)
Primers used for
amplifying GAPDH:
5’GACCCCTTCATTGACCTCAAC3’
5’CTTCTCCATGGTGGTGAAGA3’